Bioresource Technology 191 (2015) 488–495

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Advanced treatment of residual nitrogen from biologically treated coke

effluent by a microalga-mediated process using volatile fatty acids
(VFAs) under stepwise mixotrophic conditions
Byung-Gon Ryu a,b,1, Woong Kim c,1, Sung-Woon Heo c, Donghyun Kim c, Gang-Guk Choi c, Ji-Won Yang c,d,⇑
a
Environmental and Energy Program, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea
b
Decontamination and Decommissioning Research Division, Korea Atomic Energy Research Institute (KAERI), 989-111 Daedukdaero Yuseong, Daejeon 305-353, Republic of Korea
c
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea
d
Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Microalgae were used to treat the

residual NH4+-N from pretreated coke
effluent.
 Two mixotrophic cultivating modes
were used to enhance NH4+-N
removal.
 Use of VFAs in mixotrophic mode
significantly improved removal rate
of NH4+-N.
 Arctic Chlorella sp. had the highest
removal rate and FAME production.
 RDA was used to determine the
significance of factors affecting NH4+-
N removal.

a r t i c l e i n f o a b s t r a c t

Article history: This work describes the development of a microalga-mediated process for simultaneous removal of resid-
Received 28 January 2015 ual ammonium nitrogen (NH+4-N) and production of lipids from biologically treated coke effluent. Four
Received in revised form 23 March 2015 species of green algae were tested using a sequential mixotrophic process. In the first phase—CO2-sup-
Accepted 25 March 2015
plied mixotrophic condition—all microalgae assimilated NH+4-N with no evident inhibition. In second
Available online 31 March 2015
phase—volatile fatty acids (VFAs)-supplied mixotrophic condition—removal rates of NH+4-N and biomass
significantly increased. Among the microalgae used, Arctic Chlorella sp. ArM0029B had the highest rate of
Keywords:
NH+4-N removal (0.97 mg/L/h) and fatty acid production (24.9 mg/L/d) which were 3.6- and 2.1-fold
Nitrogen removal
Coke wastewater
higher than those observed under the CO2-supplied mixotrophic condition. Redundancy analysis (RDA)
Microalgae indicated that acetate and butyrate were decisive factors for increasing NH+4-N removal and fatty acid
Volatile fatty acid (VFA) production. These results demonstrate that microalgae can be used in a sequential process for treatment
Mixotrophic conditions of residual nitrogen after initial treatment of activated sludge.
Ó 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea. Tel.: +82 42 350
3964; fax: +82 42 350 3910.
E-mail address: jwyang@kaist.ac.kr (J.-W. Yang).
1
Byung-Gon Ryu and Woong Kim contributed equally to this work.

http://dx.doi.org/10.1016/j.biortech.2015.03.112
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495 489

1. Introduction
An activated sludge-based nitrification/denitrification process is
commonly used to degrade organic- and nitrogen-pollutants in the
treatment of coke wastewater (Kim et al., 2011a). This process
mainly consists of aerobic nitrification and anaerobic denitrifica-
tion. Thus, ammonia oxidizing bacteria (AOB) oxidize ammonia
(NH+3) or ammonium (NH+4) to nitrite (NO2 ), and nitrite oxidizing
bacteria (NOB) oxidize NO2 to nitrate (NO3 ). This is followed by
a heterotrophic-anaerobic reduction of NO2 or NO3 to nitrous
oxide (N2O) or dinitrogen gas (N2), with organic carbon as an elec-
tron donor (Kim et al., 2011b). Most organic pollutants are also
degraded along with these nitrogen compounds (Kim et al.,
2011a). Use of activated sludge for wastewater treatment is a reli-
able method for removal of pollutants, and is simple, economical,
and easy to scale-up. Previous pilot studies indicated that this pro-
cess is feasible (Kim et al., 2008a).
However, an unsuspected instability or sudden failure of this bio-
logical treatment process, particularly NH+4-N removal, can occur for
unknown reasons in full-scale coke wastewater treatment plants. A
major cause may be that the microbial community in the sludge is
susceptible to toxic or refractory pollutants (Kim et al., 2009). For
instance, the presence of toxic chemicals such as phenol, p-cresol,
cyanide (CN ), and thiocyanate (SCN ) in the industrial effluents
from mining, electroplating, coke, or steel-producing process can
inhibit microbial activity, particularly nitrifying- and denitrifying-
microorganisms in activated sludge. This can lead to a notable
deterioration in the efficacy of NH+4-N removal (Kim et al., 2011b,
2008b; Zhu et al., 2009). Besides, elevated levels of NH+4-N may occur
via biological oxidation of carbon–nitrogen compounds, such as
SCN , which are common in coke wastewater (Lim et al., 2008).
Because of these problems, a typical sludge-based process for treat-
ing coke effluent often fails to satisfy the strict requirements regard-
ing the level of NH+4-N in discharge (Kim et al., 2008b, 2007; Zhu
et al., 2009). Therefore, the complete remediation of residual nitro-
gen is needed after an activated sludge process for better treatment
of coke wastewater (Zhu et al., 2009).
A microalga-mediated treatment could be an efficient means to
support the removal of residual NH+4 from activated sludge because
microalgae can efficiently assimilate a variety of nitrogen com-
pounds, such as NH+4, NO3 , and NO2 , for growth under pho-
totrophic and/-or heterotrophic conditions (Pittman et al., 2011;
Ryu et al., 2014a). Most phototrophic green algae and
Cyanobacteria prefer to use NH+4 for growth, because energy is
needed following assimilation of NO2 or NO3 , which must be
reduced by intracellular reductases (Markou and Georgakakis,
2011). Furthermore, some lipid-producing (oleaginous) microalgae
can accumulate fatty acids (used for biodiesel production) at much
mixotrophic mode, in which volatile fatty acids (VFAs) were fur-
ther supplied (VFAs-supplied mixotrophic condition), was
employed to increase the rate of NH+4-N removal and produce cel-
lular lipids from the coke effluent. Redundancy analysis (RDA), a
multivariate statistical method that is an iterative analysis of reci-
procal averaging/correspondence, was used to identify environ-
mental factors that affect NH+4 removal, algal growth, and lipid
production in this system.
2. Methods
2.1. Microalgal seed cultures and sampling of coke effluent
The four species of green algae were provided by the culture
collections of the Korean Research Institute of Bioscience and
Biotechnology (Chlorella sp. ArM0029B and Ettlia sp. YC001;
KRIBB, Daejeon, Republic of Korea), the University of Texas
(Chlorella vulgaris; UTEX, Austin, TX, USA), and the Pusan
National University (Micractinium inermum F014; PNU, Pusan,
Republic of Korea). Seed cultures were gown in Tris-acetate phos-
phate (TAP) medium (pH = 7.0) with the following components:
25-mL of TAP salts, 0.375-mL of phosphate solution, 1-mL of
Hunter’s trace elements, 1-mL of glacial acetic acid, and 2.42 g of
Tris in 1 L of deionized water (Moon et al., 2013). The seeds were
cultivated in baffled hybrid flasks with vented caps that contained
100-mL of culture medium, and were maintained on a shaker
(120 rpm) at 25 °C for 5 days under continuous fluorescent light
(120 lmol/m2/s).
The biologically treated coke effluent, in which organic pollu-
tants were degraded by an activated sludge process, was obtained
from a full-scale treatment facility in an iron-producing company
in the Republic of Korea. The supernatant was collected after it
passed though the settling tank so that most solids (mainly bacter-
ial sludge) were removed. Table 1 summarizes the properties of the
coke effluent.
2.2. Cultivation of microalgae
2.2.1. CO2-supplied mixotrophic conditions
After 5 days, microalgae were separated by centrifugation
(7000 rpm for 10 min), and the pellets were transferred into the
prepared wastewater. The initial cell concentration was adjusted
to 200 mg/L dry cell weight. A lab-scale experiment for
Table 1
Characteristics of initial and biologically-treated coke wastewater used in this study.
Parameter Unit Initial coke Biologically-treated coke
wastewatera wastewaterb

higher yields than typical terrestrial oleaginous plants (Kim et al., pH 7.0 7.9
2012a,b). Thus, an alga-based wastewater treatment process may Total CODcr mgO2/ 3675.0 ± 34.4 515.0 ± 8.5
L
be an economically viable and environmentally friendly method
Soluble CODcr mgO2/ 3530.0 ± 15.1 150.0 ± 28.3
for producing commercially valuable biomass and simultaneous L
abatement of nitrogen-contaminants from wastewater (Pittman Phenol mg/L 490.0 ± 4.2 ND
et al., 2011). However, improved removal of nitrogen is needed Ammonium (NH+4) mg/L 110.7 ± 2.4 136.2 ± 2.5
because the lower rate of nitrogen removal by microalgae than Nitrate (NO3 ) mg/L 13.9 ± 0.4 2.8 ± 0.6
Nitrite (NO2 ) mg/L 3.4 ± 0.0 4.4 ± 0.0
bacteria is regarded as a drawback of microalga-based treatment Thiocyanate (SCN ) mg/L 256.3 ± 1.0 ND
(Lee et al., 2015). Fluoride (F ) mg/L 55.8 ± 0.1 43.7 ± 0.5
The purpose of the present study was to explore the feasibility Sulfate (SO24 ) mg/L 325.8 ± 4.6 2722.4 ± 0.5
of using a microalga-mediated process for treatment of residual Total solid (TS) mg/L 599.5 ± 7.8 698.0 ± 2.8
Total suspended mg/L 150.0 ± 0.0 330.0 ± 0.0
nitrogen (particularly untreated NH+4-N) from biologically treated
solid (TSS)
coke effluent in which most of the organic pollutants were already Volatile suspended mg/L 10.0 ± 0.0 35.0 ± 7.1
degraded, and to simultaneously produce lipids that can be used solid (VSS)
for biodiesel production. For the purpose, the assimilation of
ND: the element was not detected.
NH+4-N by four different strains of algae was examined under a
The actual coke wastewater before activated sludge treatment.
CO2-supplied mixotrophic condition. In a sequential manner, the b
The actual coke wastewater after activated sludge treatment.
490 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495
alga-mediated nitrogen removal was performed in a cultivating
bottle (500-mL working volume) on a shaker (150 rpm) at 25 °C
with 2% of CO2 (v/v) supplied at 0.2 vvm (volume of gas/volume of
medium/min) under continuous fluorescent light (120 lmol/m2/s).
2.2.2. VFAs-supplied mixotrophic conditions
To enhance the assimilation of NH+4 by microalgae, the inorganic
carbon source (CO2) was changed into a heterotrophic-organic car-
bon source, VFAs. The ratio of VFAs (acetic [HAc], propionic [HPr],
butyric [HBu] acids) was adjusted to 6:1:3 (by mass) because this
ratio is typical of fermented food waste in VFA platforms (Chang
et al., 2010; Fei et al., 2011). The VFA mixture was added into the
algal cultures so that the carbon nitrogen (C/N) ratio (g/g) was 40,
based on a previous study that showed maximal cell growth and
lipid accumulation for two green algae at this ratio (Ryu et al.,
2014b). Cells were incubated at 25 °C and 150 rpm, with ambient
air supplied at 0.2 vvm under continuous fluorescent light
(120 lmol/m2/s).
2.3. Analysis
2.3.1. Microbial concentration
The concentrations of volatile suspended solids (VSSs) were
analyzed as an indication of microbial growth. A 20-mL sample
was collected from the cultivating bottle and passed through a
glass microfiber filter (GF/C; Whatman, Kent, UK), which was
pre-ignited at 550 °C for 30 min in a furnace. The filtered sample
was dried at 105 °C for 2 h and then ignited at 550 °C for 30 min.
After complete ignition, the weight decrease was calculated as
amount of VSS.
2.3.2. Carbon and nitrogen pollutants
To measure the concentrations of soluble pollutants in wastewa-
ter, a 10-mL of sample was centrifuged at 8000 rpm for 10 min. The
supernatants were separated and then passed through 0.22-lm-
pore membrane filters (Sartorius Stedim Biotech, Göttingen,
Germany). The soluble chemical oxygen demand (COD), phenol,
total nitrogen (TN-N), and NH+4 were determined using water analy-
sis kits (HS-COD-MR, HS-Phenol, HS-NH3(N)-H; Humas, Daejeon,
Republic of Korea). Anions such as fluoride (F ), nitrate (NO3 ), nitrite
(NO2 ), and sulfate (SO24 ) were measured by ion chromatography
(883 Basic IC plus; Metrohm AG, Herisau, Switzerland) with an anion
column (Metrosep A Supp 5—150/4.0; Metrohm AG). SCN was
measured by addition of ferric nitrate in an acidic medium (below
pH 2) and measurement of absorbance at 460 nm with a UV–vis
spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan).
2.3.3. VFA concentrations
The concentrations of VFAs (HAc, HBu, and HPr), in the water-sol-
uble phase were measured by high-performance liquid chro-
matography (Younglin instruments, Seoul, Republic of Korea) with
a refractive index detector and an Aminex HPX-87H column (Bio-
Rad Laboratories, CA, USA). The temperatures of the column and
detector were maintained at 60 °C and room temperature, respec-
tively. A solution of 5 mM of H2SO4 (mobile eluent) was supplied
at 0.6 mL/min.
2.3.4. Chlorophyll a
Samples from a 2-mL culture sample were subjected to cen-
trifugation (12,000 rpm for 10 min) and the pellets were collected.
For extraction of chlorophyll a, dimethyl sulfoxide (DMSO) was
added to the pellet and the mixture was incubated at 60 °C for
60 min. Then absorbance at 649 and 665 nm was recorded by
UV–vis spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan),
and the chlorophyll a content was calculated as previously
described (Ryu et al., 2014b).
2.3.5. Fatty acid methyl esters (FAMEs)
The cellular lipids from microalgal cells, which were lyophilized
at 52 °C for 3 days, were extracted by a mixture of chloroform and
methanol (2:1, v/v) and then trans-esterified with methanol and sul-
furic acid by incubation at 100 °C for 20 min. To separate the
hydrophobic components (including fatty acids) from the mixture,
deionized water (DIW) was added and the mixture was vigorously
agitated. The trans-esterified fatty acids in the organic phase were
analyzed by a gas chromatograph (HP5890; Agilent, CA, USA) that
had a flame ionization detector (FID) and an INNOWAX capillary
column.
2.3.6. Redundancy analysis (RDA)
Redundancy analysis (RDA), a type of multivariate analysis
based on a linear and direct gradient-constrained ordination, is
used to interpret the relationship between variation in response
and an environmental parameter in various processes (Leps and
Smilauer, 2003). CANOCO software (version 4.5; Microcomputer,
Ithaca, NY, USA), which can test the significance of constrained
ordination models based on Mote Carlo permutation test (Leps
and Smilauer, 2003), was used for RDA. The equation used to pre-
dict the relationship of constrained ordination to multiple
multivariate regression is in Supplementary Methods.
In this study, the significance of the relationship between
environmental factors (supplied CO2 and consumed VFAs) and
their responses (removal of NH+4-N, biomass yield, FAME produc-
tion, and chlorophyll production) were analyzed. Factors were
forced to maximize the redundancy index, defined as the product
of the variance. In RDA, canonical eigenvalues correspond to the
variance in the response variables explained by the predictors
(Kim et al., 2010). The RDA results are presented by use of
CANODRAW, a part of the CANOCO software.
3. Results and discussion
3.1. Changes in organic- and nitrogen-pollutants after the activated
sludge process
Table 1 shows the physico-chemical properties of coke effluents
before (initial wastewater) and after activated sludge treatment
(biologically-treated wastewater). As expected, the biological treat-
ment process successfully degraded the soluble COD (including phe-
nol as an indicator of organic pollutants) and SCN . However, NH+4-N
levels were not decreased (even increased), despite the wastewater
being treated with a biological nitrification/denitrification step
based on the activated sludge process (Table 1). This is probably
due to inactivation of the microbial communities during the acti-
vated sludge treatment (Kim et al., 2011b; Lim et al., 2008; Ryu
et al., 2014b). When SCN is completely degraded, NH+4 accumula-
tion can occur due to the presence of aerobic–lithotrophic bacteria,
with cyanate (CNO ) and carbonyl sulfide (COS) as intermediate
compounds (Gould et al., 2012). It is possible that a decline of nitro-
gen, especially NH+4-N, never occurred because the microorganisms
involved in these reactions were repressed by the toxic organic or
inorganic chemicals and/or by secondary by-products in the coke
wastewater, such as nitrous acid from the oxidation of NH+4 (Essam
et al., 2006; Lim et al., 2008).
3.2. Removal of NH+4 by microalgae under CO2-supplied mixotrophic
conditions
Fig. 1 and Table 2 show the time course of NH+4 removal by dif-
ferent algal species under CO2-supplied mixotrophic conditions. In
general, NH+4-N is one of the most preferred N sources for pho-
totrophic microalgae, because they efficiently utilize it for growth.
 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495 491

3.3. Effects of VFAs on NH+4-N removal, chlorophyll contents, and FAME

yields

3.3.1. Removal of NH+4-N under VFAs-supplied mixotrophic condition

Fig. 1. Time course of NH+4-N removal from biologically treated coke effluent by
four microalgae under CO2-supplied mixotrophic conditions.
Previous research reported that microalgal uptake of NH+4 is greater
than that of the oxidized forms of nitrogen (NO2 and NO3 )
(Markou and Georgakakis, 2011; Perez-Garcia et al., 2011; Ryu
et al., 2014a,b). However, the coke wastewater could potentially
inhibit photosynthesis of microalgae when toxic organic pollutants
are not treated, causing poor uptake of NH+4 (Essam et al., 2006).
Fortunately, all of the microalgae tested here assimilated NH+4-N
without notable inhibition, probably due to the pre-decomposition
of toxic organic pollutants via a biological nitrification/denitrifica-
tion step based on the activated sludge process. The removal rates
were 0.21–0.27 mg/L/h, comparable with the rates previously
reported for other algae-based processes for treating wastewaters
(Fig. 1 and Table 2). Among the algae tested, Arctic Chlorella had
the best NH+4-N removal rate. Concomitant with the consumption
of NH+4-N, there were also notable increases in chlorophyll a over
time (Table 2).
However, microalga-mediated nitrogen removal is regarded as
a time-consuming process due to the high initial concentration of
NH+4 in effluent, although the removal rates reported here were
comparable to the rates from other wastewaters (Fig. 1 and
Table 2). Furthermore, there were decreases in FAME content in
all species (Table 3). This leads to the hypothesis that other toxic
components such as fluoride (F ) or sodium (Na+), which are
non-biodegradable, might inhibit an electron transfer system or
lipid synthesis in microalgae (Camargo, 2003).
After growth under CO2-supplied mixotrophic conditions, a
mixture of VFAs was added into the culture to improve the removal
of NH+4-N in batch mode, and the concentration of NH+4 was moni-
tored over time (Fig. 2). The results indicate significant increases of
NH+4-N removal in all algal cultures when the VFAs were added
(Fig. 2 and Table 2). In particular, NH+4 removal when grown with
VFAs rather than with CO2 was 3.6-fold greater in Arctic Chlorella
and 4.9-fold greater in Micractinium (Table 2). In agreement with
this finding, other research indicated that use of a subsequent het-
erotrophic mode with an additional organic carbon source allowed
efficient utilization of residual NH+4 in the medium and wastewater,
even in the presence of other microbes (Farooq et al., 2013; Ryu
et al., 2013a,b). Thus, these oleaginous strains of microalgae can
consume more nitrogen during growth by using an additional car-
bon source, and they even grow faster under heterotrophic condi-
tions than under photoautotrophic conditions. VFAs can serve as a
carbon source for the heterotrophic growth of several strains of
green microalgae (Moon et al., 2013; Perez-Garcia et al., 2011).
Fei et al. (2011) reported that the price of VFAs produced from food
waste by a VFA platform would be as low as $30/ton, much lower
than other organic carbon sources such as glucose (about $500/ton
in 2010). Therefore, use of mixotrophic growth with VFAs rather
than CO2 seems to be a feasible method for treatment of NH+4-N
in coke wastewater.
This work first demonstrated the high potential of Arctic
Chlorella for NH+4-N removal and heterotrophic consumption of
VFAs in wastewater. Ahn et al. (2012) also reported that Arctic
Chlorella had a moderate growth rate and accumulated high levels
of lipids under a broad temperature range of 4–32 °C (Ahn et al.,
2012). In a typical nitrogen removal system based on activated
sludge, seasonal failure is common because the activities of NOB
and AOB decline when the temperature is below 11 °C, resulting
in significant accumulation of oxidized nitrogen, especially NO2 ,
in colder months (Kim et al., 2006). Therefore, the eurythermy of
Arctic Chlorella makes it an ideal strain for removal of nitrogen pol-
lutants from wastewater when there are seasonal variations in
climate.
3.3.2. Changes in chlorophyll a
Chlorophyll a was also measured to assess the growth and viabil-
ity of the microalgae (Fig. 3). Under CO2-supplied mixotrophic con-
ditions, all species exhibited gradual increases in chlorophyll a, and
these increases were inversely proportional to the NH+4 levels in the

Table 2
Comparisons of the NH+4 uptake rates and chlorophyll yields of four microalgae under CO2- and VFAs-supplied mixotrophic conditions.

Algal strain Type of wastewater NH+4 uptake rate (mg/L/h) Chlorophyll yield (mg/g)g References
PAa PHb PAa PHb
Arctic Chlorella sp. ArM0029B Coke wastewaterc 0.27 ± 0.00 0.97 ± 0.00e 12.61 ± 0.33 8.50 ± 0.22 This study
Chlorella vulgaris Coke wastewaterc 0.22 ± 0.00 0.41 ± 0.04e 10.18 ± 0.02 5.64 ± 0.12
Ettlia sp. YC001 Coke wastewaterc 0.21 ± 0.01 0.70 ± 0.03e 4.89 ± 0.06 6.26 ± 0.16
Micractinium inermum F014 Coke wastewaterc 0.23 ± 0.01 1.12 ± 0.02e 13.48 ± 0.07 1.43 ± 0.07
Chlorella vulgaris Municipal wastewater 0.27 Ryu et al. (2014c)
Chlorella vulgaris Brewery wastewater 0.20 Farooq et al. (2013)
Chlorella sp. Dairy manured 0.38f Wang et al. (2010)
a
Photoautotrophic condition (PA). In this study, PA indicated the CO2-supplied mixotrophic condition.
b
Photoheterotrophic condition (PH). In this study, PH indicated the VFAs-supplied mixotrophic condition.
c
The actual coke effluent the activated sludge treatment.
d
Anaerobically digested effluent.
e
The uptake rates of NH+4 were calculated during an exponential phase.
f
The wastewater contains high levels of organic carbon including VFAs.
g
Chlorophyll yield = (Chlof Chloi)/(VSSf VSSi), where Chloi is the chlorophyll a concentration of the initial inoculums, Chlof is the chlorophyll a concentration after the
indicated duration, VSSi is the initial biomass, and VSSf is the biomass after the indicated duration.
492 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495

Table 3
Production of biomass and FAMEs under CO2- and VFAs-supplied mixotrophic conditions.

Major carbon source Biomass productivity (mg/L/d) FAMEs contents (mg/g)* FAMEs productivity (mg/L/d)
CO2 VFAs CO2 VFAs CO2 VFAs
Arctic Chlorella sp. ArM0029B 170.0 ± 7.1 380.0 ± 5.6 69.3 ± 1.9 65.7 ± 3.1 11.8 ± 0.2 24.9 ± 2.6
Chlorella vulgaris 152.5 ± 3.5 256.0 ± 5.7 57.3 ± 0.7 48.9 ± 0.4 8.7 ± 0.3 12.5 ± 0.4
Ettlia sp. YC001 168.8 ± 1.8 173.3 ± 11.3 47.6 ± 1.4 88.5 ± 0.0 8.0 ± 0.3 15.3 ± 1.0
Micractinium inermum F014 181.3 ± 2.4 370.0 ± 6.5 69.2 ± 1.2 62.6 ± 0.5 12.5 ± 0.6 23.4 ± 3.9
*
Initial FAME content was 95.3 ± 0.2 (mg/g) (Chlorella sp. ArM0029B), 91.4 ± 3.5 (mg/g) (Chlorella vulgaris), 84.6 ± 0.0 (mg/g) (Ettlia sp. YC001), and 93.9 ± 0.1 (mg/g)
(Micractinium inermum F014).

Fig. 2. Rates of NH+4-N removal by (a) Arctic Chlorella sp. ArM0029B and Chlorella vulgaris and by (b) Ettlia sp. YC001 and Micractinium inermum F014 under CO2- and VFAs-
supplied mixotrophic conditions.

Fig. 3. Changes in chlorophyll a concentration over time under CO2- and VFAs-supplied mixotrophic conditions.

wastewater. This is presumably because these microalgae utilized
the NH+4 as a nitrogen source for growth under these conditions
(Ryu et al., 2014b). In contrast, under VFAs-supplied mixotrophic
conditions, there was a steady decline in chlorophyll a as the NH+4
level declined. This indicates that the metabolism of microalgae dif-
fered under these different mixotrophic conditions. Some microal-
gae can use organic carbon as not only carbon source but also as a
source of electron donors in an electron transfer system that does
not involve chlorophyll under heterotrophic condition (Cheirsilp
and Torpee, 2012). Thus, different carbon sources appear to have dif-
ferent effects on the photosynthetic and heterotrophic metabolism
of these microalgae (Cheirsilp and Torpee, 2012).
3.3.3. Production of FAMEs and consumption of VFAs
Under VFAs-supplied mixotrophic conditions, there were
increases in the rate of NH+4-N removal, and productivities of
 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495 493

Fig. 4. Production of (a) volatile suspended solids (VSSs) and (b) FAMEs under CO2- and VFAs-supplied mixotrophic conditions.

biomass and FAMEs (Fig. 4 and Table 3). For instance, FAME pro-
ductivity (mg/L/d) was 2.1-fold higher in Arctic Chlorella and 1.9-
fold higher in Ettlia sp. than under CO2-supplied mixotrophic con-
ditions, although the FAME contents (mg/g) were not further
improved. These findings indicate that VFAs directly influenced
the increases in algal biomass and FAME yield in the biologically
treated coke effluent. Previous research also reported that an addi-
tional cultivation step to induce nitrogen starvation and maximize
the use of VFAs could improve FAME productivity, although pro-
longed nitrogen depletion may decrease FAME production due to
a loss of cell activity (Ryu et al., 2013a).
VFAs are widely available from different platforms and effluents
from anaerobic digestion of a variety of biodegradable organic
wastes, such as food, animal feces, wastewater sludge, and lipid-
extracted algae (or waste biomass of algae) (Fei et al., 2011;
Suresh et al., 2013). Moreover, cells can directly convert VFAs into
acetyl-CoA, an important intermediate in lipid synthesis (Fei et al.,
2011). VFAs therefore have great potential as an efficient and eco-
nomical carbon source to improve FAME production in oleaginous
heterotrophic microbes. Fig. 5 shows the VFAs consumed by the
Fig. 5. Consumption of acetic acid (HAc), propionic acid (HPr), and Butyric acid
four microalgae. These results demonstrate that these microalgae (HBu) by four strains of microalgae under CO2- and VFAs-supplied mixotrophic
differ in the preferences for HAc, HPr, and HBu. Therefore, conditions.
optimization may be needed to improve the complete utilization
of VFAs by microalgae, so that there are minimal residual organic
carbons in the treated effluent. a. Moreover, the rate of NH+4-N removal and FAME accumulation
Overall, these findings indicate that VFAs, an inexpensive car- were directly correlated with the levels of HAc and HBu, indicating
bon source, significantly enhanced the removal of NH+4-N and the that mixotrophic growth with VFAs had a positive effect on growth
production of lipids from biologically treated coke effluent when in the coke effluent. HAc can be easily assimilated by algal cells and
cells were grown in a sequential manner under mixotrophic condi- incorporated into acetyl-coenzyme A (acetyl-CoA), thereby provid-
tions with VFAs as a carbon source. ing carbon, energy, and reducing power through the tricarboxylic
acid (TAC) cycle. HAc is regarded as one of the most common car-
bon sources for stimulation of cellular growth in the presence of
3.4. Redundancy analysis (RDA) nitrogen for the biosynthesis of FAMEs under heterotrophic condi-
tions (Perez-Garcia et al., 2011). Similar to HAc, HBu can be readily
RDA, a multivariate ordination method, was used to identify the converted to HAc, and enter the TCA cycle, although there is
interrelationships of NH+4-N removal, chlorophyll accumulation, incomplete understanding of algal conversion of HBu to HAc
biomass accumulation, and FAME production (Fig. 6). This method (Mohan and Devi, 2012). The results also indicate that the chloro-
generates a direct gradient ordination that is related to the depen- phyll content is not always consistent with NH+4-N uptake and
dent variables (measured parameters) and independent variables FAME production because many algae shift their metabolic path-
(environmental data) (Kim et al., 2010). RDA result indicated that ways toward the biosynthesis of lipids or polysaccharides when
sum of all canonical eigenvalue was 0.962 which is a statistically grown heterotrophically, when there is abundant exogenous car-
meaningful. If a relationship is positive, the dependent variable bon (Mohan and Devi, 2012).
has a linear response to the environmental parameter. As expected, Interestingly, HPr had a linear relationship with algal growth
this analysis showed that light had a positive effect on photosyn- (biomass yield). In general, HPr rarely contributes to algal growth
thesis of microalgae because it led to an increase of chlorophyll because of its more complex structure and its modest quantity in
494 B.-G. Ryu et al. / Bioresource Technology 191 (2015) 488–495
Fig. 6. Redundancy analysis (RDA) of the correlations between environmental
variables (CO2, VFAs, HAc, HBu, and HPr) and their dependent variables (removal of
NH+4-N, biomass, chlorophyll content, FAME content, and FAME concentration). The
sum of all canonical eigenvalues is 0.962.
most anaerobically digested effluents (Fei et al., 2011; Mohan and
Devi, 2012; Moon et al., 2013). However, cells can also use HPr for
the production of oxaloacetate by an alternative pathway, and
thereby undergo growth (O’Brien and Taylor, 1977). It believed
that a proper balance between oxaloacetate and acetyl-CoA might
be necessary for the efficient oxidation of carbon sources (Perez-
Garcia et al., 2011). For instance, the accumulation of acetyl-CoA
in cells can inhibit oxaloacetate decarboxylase when the HAc is
used as a sole carbon source (Perez-Garcia et al., 2011).
The correlations of FAME content with environmental variables
were not clear in the ordination pattern. Taken together, the RDA
demonstrated that the addition of either HAc, HBu, or HPr is a deci-
sive factor for increasing the rate of NH+4-N removal and increasing
lipid production.
4. Conclusion
This work employed a microalga-mediated process for removal
of NH+4-N and production of lipids from biologically-treated coke
effluent by use of stepwise mixotrophic conditions. The activated
sludge process successfully degraded toxic organic pollutants, so
there was no subsequent inhibition of NH+4 uptake by the microal-
gae. The present findings also indicate that an additional step of
mixotrophic growth with VFAs can improve NH+4 removal and
FAME production. Taken together, these results demonstrate that
microalgae have potential for use in a sequential process for the
removal of residual nitrogen from coke effluent.
Acknowledgements
This work was supported by the Advanced Biomass R&D Center
(ABC) of Korea with a Grant from the Ministry of Science, ICT and
Future Planning (ABC-2010-0029728). This work was supported
by the National Research Foundation of Korea (NRF) grant funded
by the Korean government, Ministry of Science, ICT and Future
Planning (No. 2012M2A8A5025996).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2015.03.
112.
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